Even though there is a huge potential market for angiogenesis-related therapeutics, it is difficult to find a suitable assay to assess the effect of angiogenesis response (Staton et al., 2004). The most widely used assays are in vitro assays in which new drugs are tested in endothelial cells cultured in plastic flasks. The drawback is that it is difficult to extrapolate the observation made in an in vitro assay to model the complex process of angiogenesis actually occurring in in vivo situations (Staton et al., 2004; Taraboletti and Giavazzi, 2004). On the other hand, in vivo assays using animal models, for example mice, are costly, time-consuming and difficult for quantification (Staton et al., 2004). Thus, multiple in vitro assays are commonly employed for the identification of lead compounds. The efficacy of lead compounds on angiogenesis are subsequently validated by more than one in vivo assay. However, quantification is still an important key parameter for angiogenesis assays. Criteria for an ideal quantitative angiogenesis assay (Hasan et al., 2004) are that the assay should:                1) provide quantitative measure of structure of newly formed blood vessels;        2) provide quantitative measure of functional characters of newly formed blood vessels;        3) be able to distinguish the newly formed and pre-existing blood vessels;        4) allow long-term study;        5) be cost-effective, rapid, easy to use, reproducible; and        6) not cause any tissue damage;        
Up to now, none of the existing in vivo assays really meets these requirements (Hasan et al., 2004; Taraboletti and Giavazzi, 2004). It appears that an in vivo drug screening assay using zebrafish as model fulfill the criteria of an ideal quantitative angiogenesis assay. An in vivo zebrafish assay has the advantages and convenience of in vitro assays (high throughput comparing to in vitro assays) and in vivo assays (being an intact organism), and thus is capable to serve as bridge or filter between in vitro screening and subsequent in vivo validation (Epstein and Epstein, 2005; Goldsmith, 2004; Parng et al., 2002). An in vivo zebrafish assay would eliminate those lead compounds, due to the toxic effects, from the list identified from the in vitro assays before entering a more expensive phase of in vivo validation. An in vivo zebrafish assay has been demonstrated as a useful method for screening angiogenic drugs (reviewed by (Kidd and Weinstein, 2003)). For example, Serbedzija and his coworkers described a screening methodology which was based on the counting number of subintestinal blood vessels formed on the surface of the yolk on both side of the embryo (Serbedzija et al., 1999). Blood vessels were stained for endogenous alkaline phosphatase activity. Transgenic zebrafish lines with fluorescent blood vessels have been developed (Cross et al., 2003; Lawson and Weinstein, 2002) such that the imaging and analysis of angiogenesis can be greatly simplified. However, there is still no methodology describing fully the procedures and tools how zebrafish may be used as a quantitative tool for angiogenic drug discovery together with the corresponding toxicity test.